Protein-mediated membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. Enveloped viruses use specialized protein machinery to fuse their membrane with the membrane of host cells and deliver their genetic material for replication. In influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike is responsible for host cell attachment and membrane fusion. While structures of a subset of conformations and parts of the fusion machinery have been characterized, the states that drive the fusion process have proven to be refractory to classical structure determination. In addition, the nature of membrane deformations during fusion has largely eluded characterization. In essence, we lack a mechanistic understanding of membrane fusion and hence viral entry. This proposal focuses on determining the structural and functional interplay between hemagglutinin and membranes during fusion. A combined approach including fluorescence spectroscopy, electron cryo-tomography (ECT), small-angle X-ray scattering (SAXS) with 3-D shape reconstruction, and Hydrogen-Deuterium-exchange with mass spectrometry analysis (HD-MS) will be employed. An integrative approach is needed to reveal the mechanics of membrane fusion from the level of understanding how individual HA spikes perturb membranes to mapping the architecture of whole virion-membrane fusion complexes. In this proposal, ECT will be used to image the 3-dimensional architecture of pre-fusion, hemi-fusion and fusion pores between whole virions and target membranes. Target membranes will be chosen to test the role of lipid composition, leaflet distribution, and geometry on the fusion process. We will also use ECT to dissect the membrane-bending activity of soluble HA ectodomain and full-length HA. SAXS and HD-MS will be combined to determine the structure of the fusogenic, prehairpin HA intermediate state. The model will be validated by chemical crosslinking with mass spectrometry analysis.